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Abstract INTRODUCTION
Amiodarone (AMI) is an antiarrhythmic medication used to treat various types of irregular heartbeats (arrhythmias). It is primarily used for the treatment of ventricular arrhythmias, especially in cases where other antiarrhythmic medications have not been effective. Amiodarone has a long half-life, and this can influence the timing of dosage adjustments and potential drug interactions. Clinical testing of serum amiodarone levels is performed to monitor and optimize the therapeutic concentration of amiodarone in a patient's blood. Indication of testing include: a) monitoring therapy to ensure that the patient is receiving an effective dose for managing their arrhythmia; b) avoiding toxicity: amiodarone has a narrow therapeutic range, and levels that are too high can lead to adverse effects.
OBJECTIVES
To develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of amiodarone and its metabolite (desethylamiodarone (DEA)) and implement the test in the routine clinical diagnostic service.
METHODS
Instrument used in setting up this clinical test was a SCIEX ExionLC AD liquid chromatography system coupled to a TQ6500+ triple quadrupole mass spectrometer. ACE C18-PFP (100 x 2.1 mm, 3 um) column equipped with an in-line filter was chosen for chromatographic separation. Column oven was kept at 30 ± 1°C, while the autosampler had a thermostatted sample compartment that was maintained at 15 ± 1°C. Separation of the measurands from the matrix component was achieved using gradient elution, with solvent A: 0.1% Formic Acid in Water, and solvent B: 0.1% Formic Acid in Acetonitrile. Run time was 2.50 min. Mass spectrometer operated in the multiple reaction monitoring (MRM) mode, and positive ion polarity. Two transitions were monitored for AMI and DEA, and one for each isotopically labeled analogue used as internal standards (d4-AMI, and d4-DEA). Before LC-MS/MS analysis, proteins were precipitated from serum using acetonitrile, and diluted ten-fold using LC solvent A. Calibration standards and quality control material were made in-house from blank serum pools by adding certified reference material.
RESULTS
Method was linear between 0.1 and 20.0 mg/l for both measurands, with the correlation coefficient (r2) greater than 0.99. Within-run precision of ten replicates of 3 patient sample specimens was well below 10%, for both AMI and DEA. Between-day precision study that was monitored for two samples at mean concentrations: 1.3 mg/l and 2.0 mg/L over eleven days, generated %CVs of 5.7% and 6.3% for AMI, and 7.3% and 7.1% for DEA, respectively. Carryover was assessed for three pairs of high and low samples in triplicates, and no significant carryover was observed (<0.3% overall). Limit of quantification of of both AMI and DEA was much lower than 0.1 mg/l (Functional sensitivity, defined here as the concentration that results in a CV=20% and S/N>10) but was set to 0.1mg/l as this was medically relevant concentration. Dilution recovery was assessed by analyzing dilutions (0-, 5-, 10-, and 50-fold) of three samples within AMR (2.5, 5, 10 mg/l), and three samples above AMR (20, 35, 50mg/L) in duplicate. Assessments passed the ±15% acceptability criteria. Method comparison with the in-house HPLC method showed ~20% positive bias, while a comparison with another LC-MS/MS method a 8-10% bias was observed, with no significant interpretation changes to the test results. Evaluation of matrix effect was performed by adding the drug at a known concentration to 12 samples post-extraction (6 were non-treated and 6 were AMI-treated patient samples). Matrix effect was within total allowable error or 20%. Assessment of the sample stability showed that the concentration of both AMI and DEA is unaffected up to five freeze-thaw cycles if the serum specimen is kept at -20°C. Extract is stable in the fridge and freezer for at least 14 days.
CONCLUSION
We have established a new in-house LC-MS/MS method for rapid, simultaneous detection of amiodarone and desethylamiodarone. The improvement over the in-house HPLC method included increased specificity, shorter retention time, improved accuracy and sensitivity. The method has so far been robust and free of interferences. |
= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
